Essential oil of Blue tansy obtained from the flowers and leaves of Tanacetum annuum (Asteraceae) by steam distillation

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Tanacetum annuum

IUPAC Name:

Essential oil of Tanacetum annuum

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK, (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler))
- Number of animals: Multiple
- Characteristics of donor animals (e.g. age, sex, weight): approximately 3 kg and approximately 56 days old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: same day
- indication of any existing defects or lesions in ocular tissue samples: The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL to the cornea
- Concentration (if solution): unchanged

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected. Eyelids were carefully excised and the integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined for damage of cornea with the use of the Haag-Streit BQ 900 (Switzerland) microscope, An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are or smaller then 0.5.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip and examined again for damage. The temperature of the chambers was at 32 ±1.5 °C.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Benzalkonium chloride 5 v/v

APPLICATION DOSE AND EXPOSURE TIME: 0.3 mL was applied for 10 seconds

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds of exposure, the surface of the cornea was rinsed with 20 mL isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated with the most densely opacified areas for scoring
- Damage to epithelium based on fluorescein retention: calculated at the 30 minute time interval only
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Percentage corneal swelling was assessed from corneal thickness measurements.
- Macroscopic morphological damage to the surface: evaluated with Haag-Streit BQ 900 (Switzerland) microscope

DECISION CRITERIA:
Once each endpoint had been established, ICE classes were determined based on a predetermined range. The overall in vitro irritancy classification of the test item was determined by using all the
results applied it to ICE classification criteria. A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

0, 30, 75, 120, 240 minutes

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

30 minutes

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

30 minutes

Value:

4.46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

75 minutes

Value:

9.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

120 minutes

Value:

8.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

180 minutes

Value:

7.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

240 minutes

Value:

10.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, valid positive and negative controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean corneal opacity 0, mean Fluorescein retention 0, Mean corneal swelling up to 3.70%; 3x ICE class I)
- Acceptance criteria met for positive control: yes (mean corneal opacity up to 4.0, mean Fluorescein retention 2.7, Mean corneal swelling up to 39.60%; 3x ICE class IV)

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Remarks:

based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Under the conditions of this test, Blue Tansy oil does not need to be classified for eye irritation, according to the criteria laid down in Annex I of 1272/2008/EC (CLP).

Executive summary:

In an isolated chicken eye test, performed according to the 438 OECD Guideline under GLP conditions, Blue Tansy oil was tested to evaluate the eye hazard potential of the substance. 0.3 mL of the test item was applied to the cornea of each of 3 chicken enucleated eyes and after 10 seconds the corneas were rinsed with isotonic saline. After the exposure, toxic effects were measured by qualitative assessment of opacity (0.3, ICE class I), fluorescein retention (0.5 ICE class I), and corneal swelling (10.89% ICE class II). Valid positive (overall ICE class IV) and negative (overall ICE class I) controls were included. According to the overall in vitro irritancy criteria, the ICE classes combined for teat item lead to result ‘no Category’. Therefore under the conditions of this test, the test item does not need to be classified for eye irritation, according to the criteria laid down in Annex I of 1272/2008/EC (CLP).